In the last 30 years, the incidence of birth defects affecting external genitalia has more than doubled. The most common of these defects, hypospadias, occurs when the urethral tube opens on the ventral side of the penis. In severe cases, boys are born with feminized genitalia. Rates of hypospadias are alarmingly high occurring in 1 in 125 male births. Embryonic exposure to environmental endocrine disrupting chemicals (EDCs) is hypothesized to be a primary cause of hypospadias. In fact, EDC exposure of mothers and presence of EDCs in the placenta have both been correlated with incidence of hypospadias in sons. Currently there are no preventative therapies for hypospadias. However, we recently discovered that supplementing EDC-exposed mice with vitamin E reduces the severity of hypospadias in their sons. This project investigates the developmental and molecular mechanisms producing vitamin E's ameliorative effects, and has two multifaceted specific aims 1) to investigate Vitamin E's effects on morphogenesis including cell invasion, proliferation, and apoptosis (cell death), 2) to investigate Vitamin E's ameliorative effects on quantitative and spatial patterns of gene expression. To investigate how Vitamin E influences cellular invasion, shhGFPcre and Rosa26-LacZ (R26R) reporter transgenic mice will be used to visualize the urethral epithelium relative to the surrounding mesenchyme after treatment by oral gavage of pregnant dams with vinclozolin, vinclozolin plus vitamin E, or vehicle alone. Using the same experimental design I will investigate vitamin E's effects on cell proliferation and apoptosis in non-transgenic mice. To measure cell proliferation I will label mitotic cells in the S and G2/M phases of the cell cycle using immunohistochemistry. Apoptosis will be measured using Lysotracker Red a lysosomotropic dye that fluorescently labels apoptotic cells. To investigate the effects vitamin E on quantitative and spatial patterns of gene expression I will again use the same experimental designs as above but will run a microarray analysis on the genital tubercles of embryos and then map the tissue specific patterns of differentially expressed genes onto tubercles of age matched embryos exposed to the same treatments using in situ hybridization. My long term objective for this work is to develop a nutritive supplement that reduces the incidence and severity of hypospadias. My research is directly relevant to the mission of both the NIH and NIEHS in that its specific aims are designed to enhance health, and reduce burdens of illness and disability and to understand how the environment influences the development of human disease.